Evaluation of Chitosan Derivatives Modified Mesoporous Silica Nanoparticles as Delivery Carrier.

Chitosan is a non-toxic biological material, but chitosan is insoluble in water, which hinders the development and utilization of chitosan. Chitosan derivatives N-2-Hydroxypropyl trimethyl ammonium chloride (N-2-HACC) and carboxymethyl chitosan (CMCS) with good water solubility were synthesized by our laboratory. In this study, we synthesized mesoporous SiO2 nanoparticles by the emulsion, and then the mesoporous SiO2 nanoparticles were modified with γ-aminopropyltriethoxysilane to synthesize aminated mesoporous SiO2 nanoparticles; CMCS and N-2-HACC was used to cross-link the aminated mesoporous SiO2 nanoparticles to construct SiO2@CMCS-N-2-HACC nanoparticles. Because the aminated mesoporous SiO2 nanoparticles with positively charged can react with the mucous membranes, the virus enters the body mainly through mucous membranes, so Newcastle disease virus (NDV) was selected as the model drug to evaluate the performance of the SiO2@CMCS-N-2-HACC nanoparticles. We prepared the SiO2@CMCS-N-2-HACC nanoparticles loaded with inactivated NDV (NDV/SiO2@CMCS-N-2-HACC). The SiO2@CMCS-N-2-HACC nanoparticles as delivery carrier had high loading capacity, low cytotoxicity, good acid resistance and bile resistance and enteric solubility, and the structure of NDV protein encapsulated in the nano vaccine was not destroyed. In addition, the SiO2@CMCS-N-2-HACC nanoparticles could sustain slowly released NDV. Therefore, the SiO2@CMCS-N-2-HACC nanoparticles have the potential to be served as delivery vehicle for vaccine and/or drug.

Adjuvants and delivery systems based on polymeric nanoparticles for mucosal vaccines.

Most pathogens enter the body through mucosal surfaces. Therefore, vaccination through the mucosal route can greatly enhance the mucosal immune response. Vaccination via the mucosal surface is the most effective way to trigger a protective mucosal immune response, but the vast majority of vaccines used are administered by injection. Strategies to enhance the mucosal immunity have been developed by using vaccine adjuvants, delivery systems, bacterial or viral vectors, and DNA vaccines. Appropriate vaccine adjuvants and drug delivery systems can improve the immunogenicity of antigens, induce a stronger immune response, and reduce the vaccine dose and production cost. In recent years, many studies have focused on finding safe and effective vaccine adjuvants and drug delivery systems to formulate the mucosal vaccines for solving the above problems. Great progress has also been made in vaccine adjuvants and drug delivery systems based on biodegradable polymer nanoparticles. In this paper, the research progress of the mucosal vaccine and its related adjuvants and drug delivery systems in recent years was reviewed, and the application of polymers as adjuvants and drug delivery system in vaccine was prospected. This review provides a fundamental knowledge for the application of biodegradable polymer nanoparticles as adjuvants and carriers in mucosal vaccines and shows great application prospects.

Sex determination by SRY PCR and sequencing of Tasmanian devil facial tumour cell lines reveals non-allograft transmission.

Devil facial tumour disease (DFTD) is an infectious tumour disease and was hypothesised to be transmitted by allograft during biting based on two cytogenetic findings of DFTD tumours in 2006. It was then believed that DFTD tumours were originally from a female devil. In this study the devil sex-determining region Y (SRY) gene was PCR amplified and sequenced, and six pairs of devil SRY PCR primers were used for detection of devil SRY gene fragments in purified DFTD tumour cell lines. Using three pairs of devil SRY PCR primers, devil SRY gene sequence was detected by PCR and sequencing in genomic DNA of DFTD tumour cell lines from six male devils, but not from six female devils. Four out of six DFTD tumour cell lines from male devils contained nucleotides 288-482 of the devil SRY gene, and another two DFTD tumour cell lines contained nucleotides 381-577 and 493-708 of the gene, respectively. These results indicate that the different portions of the SRY gene in the DFTD tumours of the male devils were originally from the male hosts, rejecting the currently believed DFTD allograft transmission theory. The reasons why DFTD transmission was incorrectly defined as allograft are discussed.

Effects of reticuloendotheliosis virus infection on cytokine production in SPF chickens.

Infection with reticuloendotheliosis virus (REV), a gammaretrovirus in the Retroviridae family, can result in immunosuppression and subsequent increased susceptibility to secondary infections. The effects of REV infection on expression of mRNA for cytokine genes in chickens have not been completely elucidated. In this study, using multiplex branched DNA (bDNA) technology, we identified molecular mediators that participated in the regulation of the immune response during REV infection in chickens. Cytokine and chemokine mRNA expression levels were evaluated in the peripheral blood mononuclear cells (PBMCs). Expression levels of interleukin (IL)-4, IL-10, IL-13 and tumor necrosis factor (TNF)-α were significantly up-regulated while interferon (IFN)-α, IFN-ß, IFN-γ, IL-1ß, IL-2, IL-3, IL-15, IL-17F, IL-18 and colony-stimulating factor (CSF)-1 were markedly decreased in PBMCs at all stages of infection. Compared with controls, REV infected chickens showed greater expression levels of IL-8 in PBMCs 21 and 28 days post infection. In addition, REV regulates host immunity as a suppressor of T cell proliferative responses. The results in this study will help us to understand the host immune response to virus pathogens.

Complete genome sequence of the first Chinese virulent infectious laryngotracheitis virus.

Infectious laryngotracheitis (ILT) is an acute respiratory disease caused by infectious laryngotracheitis virus (ILTV). The complete genome sequences of five attenuated ILTV vaccine strains and six virulent ILTV strains as well as two Australian ILTV field strains have been published in Australia and the USA so far. To provide the complete genome sequence information of ILTVs from different geographic regions, the whole genome of ILTV LJS09 isolated in China was sequenced. The genome of ILTV LJS09 was 153,201 bp in length, and contained 79 ORFs. Most of the ORFs had high sequence identity with homologous ORFs of reference strains. There was a large fragment deletion within the noncoding region of unique long region (UL) of ILTV LJS09 compared with SA2 and A20 strains. Though the origin binding protein of ILTV LJS09 existed, there was no AT-rich region in strain LJS09. Alignments of the amino acid sequences revealed seven mutations at amino acids 71 (Arg → Lys), 116 (Ala → Val), 207 (Thr → Ile) and 644 (Thr → Ile) on glycoprotein B, 155 (Phe → Ser) and 376 (Arg → His) on glycoprotein D and 8 (Gln→Pro) on glycoprotein L of ILTV LJS09 compared to those of virulent strain (USDA) as ILTV LJS09 did not grow on chicken embryo fibroblasts, suggesting the role of the key seven amino acids in determination of the cell tropism of ILTV LJS09. This is the first complete genome sequence of the virulent strain of ILTV in Asia using the conventional PCR method, which will help to facilitate the future molecular biological research of ILTVs.

Detection of infectious laryngotracheitis virus by real-time PCR in naturally and experimentally infected chickens.

Infectious laryngotracheitis (ILT) is an acute, highly contagious upper-respiratory infectious disease of chickens. In this study, a real-time PCR method was developed for fast and accurate detection and quantitation of ILTV DNA of chickens experimentally infected with ILTV strain LJS09 and naturally infected chickens. The detection lower limit of the assay was 10 copies of DNA. There were no cross reactions with the DNA and RNA of infectious bursal disease virus, chicken anemia virus, reticuloendotheliosis virus, avian reovirus, Newcastle disease virus, and Marek's disease virus. The real-time PCR was reproducible as the coefficients of variation of reproducibility of the intra-assay and the inter-assay were less than 2%. The real-time PCR was used to detect the levels of the ILTV DNA in the tissues of specific pathogen free (SPF) chickens infected with ILTV at different times post infection. ILTV DNA was detected by real-time PCR in the heart, liver, spleen, lung, kidney, larynx, tongue, thymus, glandular stomach, duodenum, pancreatic gland, small intestine, large intestine, cecum, cecal tonsil, bursa of Fabricius, and brain of chickens in the infection group and the contact-exposure group. The sensitivity, specificity, and reproducibility of the ILTV real-time PCR assay revealed its suitability for detection and quantitation of ILTV in the samples from clinically and experimentally ILTV infected chickens.

Avirulent Marek's disease virus type 1 strain 814 vectored vaccine expressing avian influenza (AI) virus H5 haemagglutinin induced better protection than turkey herpesvirus vectored AI vaccine.

BACKGROUND: Herpesvirus of turkey (HVT) as a vector to express the haemagglutinin (HA) of avian influenza virus (AIV) H5 was developed and its protection against lethal Marek's disease virus (MDV) and highly pathogenic AIV (HPAIV) challenges was evaluated previously. It is well-known that avirulemt MDV type 1 vaccines are more effective than HVT in prevention of lethal MDV infection. To further increase protective efficacy against HPAIV and lethal MDV, a recombinant MDV type 1 strain 814 was developed to express HA gene of HPAIV H5N1. METHODOLOGY/PRINCIPAL FINDINGS: A recombinant MDV-1 strain 814 expressing HA gene of HPAIV H5N1 virus A/goose/Guangdong/3/96 at the US2 site (rMDV-HA) was developed under the control of a human CMV immediate-early promoter. The HA expression in the rMDV-HA was tested by immunofluorescence and Western blot analyses, and in vitro and in vivo growth properties of rMDV-HA were also analyzed. Furthermore, we evaluated and compared the protective immunity of rMDV-HA and previously constructed rHVT-HA against HPAIV and lethal MDV. Vaccination of chickens with rMDV-HA induced 80% protection against HPAIV, which was better than the protection rate by rHVT-HA (66.7%). In the animal study with MDV challenge, chickens immunized with rMDV-HA were completely protected against virulent MDV strain J-1 whereas rHVT-HA only induced 80% protection with the same challenge dose. CONCLUSIONS/SIGNIFICANCE: The rMDV-HA vaccine was more effective than rHVT-HA vaccine for protection against lethal MDV and HPAIV challenges. Therefore, avirulent MDV type 1 vaccine is a better vector than HVT for development of a recombinant live virus vaccine against virulent MDV and HPAIV in poultry.

Identification of a conserved B-cell epitope on reticuloendotheliosis virus envelope protein by screening a phage-displayed random peptide library.

BACKGROUND: The gp90 protein of avian reticuloendotheliosis-associated virus (REV-A) is an important envelope glycoprotein, which is responsible for inducing protective antibody immune responses in animals. B-cell epitopes on the gp90 protein of REV have not been well studied and reported. METHODS AND RESULTS: This study describes the identification of a linear B-cell epitope on the gp90 protein by screening a phage-displayed 12-mer random peptide library with the neutralizing monoclonal antibody (mAb) A9E8 directed against the gp90. The mAb A9E8 recognized phages displaying peptides with the consensus motif SVQYHPL. Amino acid sequence of the motif exactly matched (213)SVQYHPL(219) of the gp90. Further identification of the displayed B cell epitope was conducted using a set of truncated peptides expressed as GST fusion proteins and the Western blot results indicated that (213)SVQYHPL(219) was the minimal determinant of the linear B cell epitope recognized by the mAb A9E8. Moreover, an eight amino acid peptide SVQYHPLA was proven to be the minimal unit of the epitope with the maximal binding activity to mAb A9E8. The REV-A-positive chicken serum reacted with the minimal linear epitopes in Western blot, revealing the importance of the eight amino acids of the epitope in antibody-epitope binding activity. Furthermore, we found that the epitope is a common motif shared among REV-A and other members of REV group. CONCLUSIONS AND SIGNIFICANCE: We identified (213)SVQYHPL(219) as a gp90-specific linear B-cell epitope recognized by the neutralizing mAb A9E8. The results in this study may have potential applications in development of diagnostic techniques and epitope-based marker vaccines against REV-A and other viruses of the REV group.

Preparation and efficacy of a live newcastle disease virus vaccine encapsulated in chitosan nanoparticles.

BACKGROUND: Newcastle disease (ND) is a highly contagious viral disease of poultry caused by pathogenic strains of the Newcastle disease virus (NDV). Live NDV vaccines are administered by drinking water, eyedrops or coarse aerosol spray. To further enhance mucosal immune responses, chitosan nanoparticles were developed for the mucosal delivery of a live NDV vaccine. METHODOLOGY/PRINCIPAL FINDINGS: A lentogenic live-virus vaccine (strain LaSota) against NDV encapsulated in chitosan nanoparticles were developed using an ionic crosslinking method. Chitosan nanoparticles containing the lentogenic live-virus vaccine against NDV (NDV-CS-NPs) were produced with good morphology, high stability, a mean diameter of 371.1 nm, an encapsulation rate of 77% and a zeta potential of +2.84 mV. The Western blotting analysis showed that NDV structural proteins were detected in NDV-CS-NPs. The virus release assay results of NDV-CS-NPs indicated that NDV was released from NDV-CS-NPs. Chickens immunized orally or intranasally with NDV-CS-NPs were fully protected whereas one out of five chickens immunized with the LaSota live NDV vaccine and three out of five chickens immunized with the inactivated NDV vaccine were dead after challenge with the highly virulent NDV strain F48E9. CONCLUSIONS/SIGNIFICANCE: NDV-CS-NPs induced better protection of immunized specific pathogen free chickens compared to the live NDV vaccine strain LaSota and the inactivated NDV vaccine. This study lays a foundation for the further development of mucosal vaccines and drugs encapsulated in chitosan nanoparticles.

Expression of HA of HPAI H5N1 virus at US2 gene insertion site of turkey herpesvirus induced better protection than that at US10 gene insertion site.

Herpesvirus of turkey (HVT) is being widely used as a vector for development of recombinant vaccines and US2 and US10 genes are often chosen as insertion sites for targeted gene expression. However, the different effects of the two genes for generation of recombinant HVT vaccines were unknown. In order to compare the effects of inserted genes in the two sites on the efficacy of the recombinant vaccines, host-protective haemagglutinin (HA) gene of the highly pathogenic avian influenza virus (HPAIV) H5N1 was inserted into either US2 or US10 gene locus of the HVT. The resulting US2 (rHVT-US2-HA) or US10 (rHVT-US10-HA) recombinant HVT viruses were used to infect chicken embryo fibroblasts. Plaques and the growth kinetics of rHVT-US2-HA-infected chicken embryo fibroblasts were similar to those of parental HVT whereas rHVT-US10-HA infected chicken embryo fibroblasts had different growth kinetics and plaque formation. The viremia levels in rHVT-US10-HA virus-infected chickens were significantly lower than those of rHVT-US2-HA group on 28 days post infection. The vaccine efficacy of the two recombinant viruses against H5N1 HPAIV and virulent Marek's disease virus was also evaluated in 1-day-old vaccinated chickens. rHVT-US2-HA-vaccinated chickens were better protected with reduced mortality than rHVT-US10-HA-vaccinated animals following HPAIV challenge. Furthermore, the overall hemaglutination inhibition antibody titers of rHVT-US2-HA-vaccinated chickens were higher than those of rHVT-US10-HA-vaccinated chickens. Protection levels against Marek's disease virus challenge following vaccination with either rHVT-US2-HA or rHVT-US10-HA, however, were similar to those of the parental HVT virus. These results, for the first time, indicate that US2 gene provides a favorable foreign gene insertion site for generation of recombinant HVT vaccines.

Humoral immune responses in brushtail possums (Trichosurus vulpecula) induced by bacterial ghosts expressing possum zona pellucida 3 protein.

The introduced common brushtail possum (Trichosurus vulpecula) is a major pest in New Zealand and immunocontraceptive vaccines are being developed for biocontrol of possum populations, with bacterial ghosts (BGs) being evaluated as a means of oral delivery. Recombinant BGs expressing possum zona pellucida 3 protein (ZP3) as an L' membrane-anchored protein (ZP3-L') or as an S-layer SbsA-fusion protein (MBP-SbsA-ZP3) were produced by the expression of the cloned bacteriophage phiX174 lysis gene E in E. coli NM522. The humoral immune responses of possums immunised with BGs expressing possum ZP3 were investigated following oral, intranasal/conjunctival, parenteral, and intraduodenal administration to evaluate the BG-ZP3 system for possum fertility control. Antibodies to possum ZP3 were detected in the serum, oviduct secretions, and follicular fluid of immunised animals. Intranasal/conjunctival immunisation elicited reliable antibody immune response in serum and at a key effector site, the ovarian follicular fluid. Intraduodenal administration of possum ZP3 BG vaccine as a priming immunisation elicited significant systemic immune responses, but oral immunisation did not, indicating that protection of BG vaccines from degradation by gastric acidity would enhance the effectiveness of orally delivered vaccines. The detection of antibodies at elevated levels at target sites in the reproductive tract following mucosal delivery demonstrates, for the first time, the potential of BGs as an effective system for vaccine delivery to wild animals, and intranasal/conjunctival immunisation as a promising means for delivery of immunocontraceptive vaccines to wild animals.

Identification and evaluation of an infertility-associated ZP3 epitope from the marsupial brushtail possum (Trichosurus vulpecula).

Immunologically based fertility control vaccines against zona pellucida (ZP) proteins are being developed in New Zealand for biocontrol of the brushtail possum (Trichosurus vulpecula), an introduced Australian marsupial pest. We have shown that immunization of female possums with recombinant possum ZP3 protein (rZP3) reduced fertility by 79%. To enhance the specificity of possum immunocontraceptive vaccines, B-cell epitopes on possum ZP3 protein were mapped using sera of female possums immunized with possum rZP3 and subjected to a fertility trial. The amino acid sequence of the full-length possum ZP3 protein was used to synthesize a complete set of 83 (12-mer) biotinylated peptides each with an overlap of five amino acids with the neighboring peptides. The peptides were used in a modified enzyme-linked immunosorbent assay (ELISA) to identify continuous epitopes recognized by antibodies in the sera of possums immunized with possum rZP3. Sixteen epitopes were identified on the possum ZP3 protein. Comparison of the ELISA binding patterns of these peptides to antibodies in the individual sera with the fertility status of rZP3-immunized possums identified only one epitope (amino acids 156-172) to be associated with infertility. However, female possums immunized with this epitope showed no significant reduction in fertility. The possible reasons for the failure of this potential infertility epitope are discussed.

Bacterial ghosts as a delivery system for zona pellucida-2 fertility control vaccines for brushtail possums (Trichosurus vulpecula).

The introduced brushtail possum is a serious pest in New Zealand and there is much interest in the development of an immunocontraceptive vaccine for population control. Immunisation of female possums against recombinant possum zona pellucida protein-2 (ZP2) is known to reduce embryo production by 72-75% but successful development of fertility control will depend on a delivery system that is effective for field use. Bacterial ghost vaccine technology is a promising system to formulate a non-living vaccine for bait or aerosol delivery. The N-terminal (amino acid residues 41-316, ZP2N) and C-terminal (amino acid residues 308-636, ZP2C) regions of possum ZP2 were fused to maltose-binding protein and expressed in the periplasmic space of Escherichia coli NM522 bacterial ghosts. Female possums (n=20 per treatment group) were immunised with 20mg of either plain ghosts, ZP2N ghosts, or ZP2C ghosts in phosphate-buffered saline applied to the nostrils and eyes (nasal/conjunctival mucosa) at weeks 0, 2 and 4. Effects of immunisation on fertility were assessed following superovulation and artificial insemination. Both constructs evoked humoral (antibody) and cell-mediated immune responses in possums and significantly fewer eggs were fertilised in females immunised against ZP2C ghosts. Results in this study indicate that bacterial ghosts containing possum ZP antigens can reduce possum fertility when delivered by mucosal immunisation and offer a promising delivery system for fertility control of wild possum populations.

Immunogenicity and contraceptive potential of three infertility-relevant zona pellucida 2 epitopes in the marsupial brushtail possum (Trichosurus vulpecula).

In a previous study, three infertility-relevant epitopes of possum ZP2 (Pep12 (amino acids 111-125), Pep31 (amino acids 301-315), and Pep44 (amino acids 431-445)) were identified using sera from possums (Trichosurus vulpecula) immunized with recombinant possum zona pellucida 2 (ZP2) constructs, and a synthetic peptide library of possum ZP2 protein. In this study, the three peptides were conjugated to keyhole limpet hemocyanin and 300 mug of each conjugated peptide were administered subcutaneously to female possums (n = 20 per peptide) in complete Freund's adjuvant. Immunogen doses were repeated 3 and 6 weeks later using incomplete Freund's adjuvant. Control animals were immunized with either phosphate-buffered saline only (n = 10) or 300 mug keyhole limpet hemocyanin (n = 10), administered with the same adjuvants. Serum antibodies from animals immunized against these three epitopes bound to the corresponding possum ZP2 peptides, recombinant possum ZP2 protein constructs, and native zona. Possum fertility was assessed following superovulation and artificial insemination. Peptides Pep12 and Pep31 had no significant effects on fertility parameters (P > 0.05). However, animals immunized with Pep44 had lower egg fertilization rates (immunized 19.5% versus control 60.5%, P < 0.05) and produced significantly fewer embryos than control animals (immunized 0.5 embryos versus control 2.4 embryos, P < 0.05). The number of Pep44-immunized females that produced embryos was reduced by 64%. Identification and characterization of possum infertility-relevant epitopes on possum ZP2 protein will assist development of safe, humane, and possum-specific immunocontraceptive vaccines for controlling the introduced possums in New Zealand.

Mapping of B cell epitopes on the zona pellucida 2 protein of a marsupial, the brushtail possum (Trichosurus vulpecula).

Immunocontraceptive vaccines against zona pellucida (ZP) proteins are being developed for brushtail possum (Trichosurus vulpecula) management in New Zealand. Mapping of B cell epitopes on the ZP2 protein of possums was undertaken in this study to define the antigenic regions that may be crucial to sperm-egg binding. The amino acid sequence of the full-length possum ZP2 protein (712 amino acids) was used to synthesize a complete set of 71 (15-mer) biotinylated peptides with an offset of five amino acids. The peptides were used in a modified enzyme-linked immunosorbent assay (ELISA) to identify continuous epitopes recognized by antibodies in the sera of possums immunized with recombinant possum ZP2 (rZP2) constructs. Seventeen continuous epitopes were located on possum ZP2 protein. Comparisons of the peptide binding pattern of antibodies in individual sera with the fertility status of the same immunized possums revealed three significant infertility-relevant peptide epitopes (amino acids 111-125, 301-315, and 431-445). One of these (amino acids 431-445) bound to possum spermatozoa from the caudal epididymis. The implications of these findings for developing immunocontraceptive vaccines for possum control are discussed.

Mapping of conformational epitopes on capsid protein VP2 of infectious bursal disease virus by fd-tet phage display.

Conformational epitopes on VP2 protein of infectious bursal disease virus (IBDV) were mapped using fd-tet phage display. A gene-targeted phage display library was made using DNA fragments ranging approximately from 80 to 400 bp of the hypervariable region of the VP2 gene of IBDV strain 002-73, as neutralizing monoclonal antibodies against the VP2 protein recognize VP2 conformation-dependent epitopes within the hypervariable region. The phages were selected using immobilized monoclonal antibodies. Epitopes on five phages selected with monoclonal antibody 17-82 were located between amino acids 211 and 344. A constructed phage containing amino acids from 204 to 344 strongly reacted with monoclonal antibodies. Compared to that of the constructed phage, the binding of monoclonal antibodies to the five selected phages was dramatically reduced when several amino acids at either terminus or both termini were absent. The binding of a phage, with conversion of the first hydrophilic region into a hydrophobic region as a result of a chance frameshift mutation from amino acids 214 to 225, dropped sharply. It indicates that conformational epitopes may be up to 423 bp long and the commonly suggested fragments of 50-300 bp for making gene-targeted phage display libraries are not long enough to cover the conformational epitopes. This technique can be used to identify the minimum length of the conformational epitopes for developing recombinant vaccines and specific diagnostic tests.

Identification of crucial residues of conformational epitopes on VP2 protein of infectious bursal disease virus by phage display.

A new method for identifying epitopes in viral proteins expressed by filamentous phage has been developed. Filamentous phage fUSE 1 containing the variable region of the VP2 gene of infectious bursal disease virus (IBDV) strain 002-73 was constructed. Neutralizing monoclonal antibodies 17-82 and 33-10 raised against VP2 protein were used to bind phage containing the original variable region of VP2. The phage bound to monoclonal antibodies, were removed by protein G Sepharose and the unbound phage (escape mutants) were isolated for sequencing to locate the mutations. The crucial amino acid residues for conformational neutralizing epitopes recognized by the monoclonal antibodies were located in the first main hydrophilic region (amino acids from 210 to 225) and the central region of the variable region of VP2. The amino acid residues on both ends of the variable region of VP2 affected considerably the binding of monoclonal antibodies. This technique might be useful for selecting escape mutants of phage displaying the original antigenic regions of other viruses to define the crucial amino acid residues of their conformational epitopes, especially viruses that cannot be grown in cell cultures.